Bimodal interaction of coatomer with the p24 family of putative cargo receptors.

Cytoplasmic domains of members of the p24 family of putative cargo receptors were shown to bind to coatomer, the coat protein of COPI-coated transport vesicles. Domains that contained dilysine endoplasmic reticulum retrieval signals bound the alpha-, beta'-, and epsilon-COP subunits of coatomer, whereas other p24 domains bound the beta-, gamma-, and zeta-COP subunits and required a phenylalanine-containing motif. Transit of a CD8-p24 chimera from the endoplasmic reticulum through the Golgi complex was slowed when the phenylalanine motif was mutated, suggesting that this motif may function as an anterograde transport signal. The either-or bimodal binding of coatomer to p24 tails suggests models for how coatomer can potentially package retrograde-directed and anterograde-directed cargo into distinct COPI-coated vesicles.

An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.

We have isolated a major integral membrane protein from Golgi-derived coatomer-coated vesicles. This 24-kDa protein, p24, defines a family of integral membrane proteins with homologs present in yeast and humans. In addition to sequence similarity, all p24 family members contain a motif with the characteristic heptad repeats found in coiled coils. When the yeast p24 isoform, yp24A, is knocked out in a strain defective for vesicle fusion, a dramatic reduction in the accumulation of transport vesicles is observed. Together, these results indicate a role for this protein family in the budding of coatamer-coated and other species of coated vesicles.

Thermodynamics of cyclophilin catalyzed peptidyl-prolyl isomerization by NMR spectroscopy.

One-dimensional nmr exchange spectroscopy was carried out to determine thermodynamic parameters of cyclophilin-induced cis-trans isomerization of succinyl-Ala-Phe-Pro-Phe-p-nitroanilide. Rate measurements were possible at physiological temperatures. The kc/Km of rat cyclophilin was found to be 12.8 (+/- 0.5) s-1 microM-1 at 37 degrees C, intermediate to previously reported values that used a coupled enzyme assay extrapolated to this temperature. Activation energies (delta G not equal to) for the uncatalyzed and catalyzed reaction at 37 degrees C were found to be 19.7 and 17.1 kcal/mol, respectively, and were primarily due to an enthalpic barrier.

The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein.

The small GTP-binding protein, ADP-ribosylation factor (ARF), has previously been shown to mediate the binding to Golgi membranes of the coatomer of non-cathrin-coated (COP-coated) vesicles. We now report that ARF is also required for the binding of the AP-1 adaptor protein of clathrin-coated vesicles to Golgi membranes. The binding of coat proteins from both clathrin- and COP-coated vesicles requires an additional Golgi membrane-associated factor. These results suggest that a mechanistic similarity underlies diverse types of vesicle coats.

Cyclophilins: a new family of proteins involved in intracellular folding.

Proteins of the cyclophilin family display two intriguing properties. On the one hand, they are the intracellular receptors for the immunosuppressive drug cyclosporin A (CsA); on the other hand, they function in vitro as enzymes that catalyse slow steps in protein folding. A dissection of the role of CsA in mediating immunosuppression, together with recent studies on the biology of cyclophilins in the absence of this ligand, is providing fundamental insight into the cellular function of this protein family.

Genetic dissection of cyclophilin function. Saturation mutagenesis of the Drosophila cyclophilin homolog ninaA.

Cyclophilins, the intracellular receptors for the widely used immunosuppressant cyclosporin A have been found to be peptidyl-prolyl cis/trans isomerases and have been implicated in intracellular protein folding and trafficking. The Drosophila ninaA gene encodes a photoreceptor-specific cyclophilin homolog involved in rhodopsin biogenesis. ninaA mutants have a 90% reduction in the levels of Rh1 rhodopsin. To gain insight into the role of cyclophilins in vivo, we carried out a genetic screen designed to identify functionally important regions in the ninaA protein. Over 700,000 mutagenized flies were screened for a visible ninaA phenotype and 70 independent mutations in ninaA were isolated and characterized. These mutations provide a detailed dissection of the structure/function relationships in cyclophilin. We also show that mammalian cyclophilins engineered to contain missense mutations found in two temperature-sensitive ninaA alleles display temperature-sensitive prolyl cis/trans isomerase activity.

The cyclophilin homolog ninaA is required in the secretory pathway.

In Drosophila, the major rhodopsin Rh1 is synthesized in endoplasmic reticulum (ER)-bound ribosomes of the R1-R6 photoreceptor cells and is then transported to the rhabdomeres where it functions in phototransduction. Mutations in the cyclophilin homolog ninaA lead to a 90% reduction in Rh1 opsin. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We now show that mutations in the ninaA gene severely inhibit opsin transport from the ER, leading to dramatic accumulations of ER cisternae in the photoreceptor cells. These results demonstrate that ninaA functions in the ER. Interestingly, ninaA and Rh1 also colocalize to secretory vesicles, suggesting that Rh1 may require ninaA as it travels through the distal compartments of the secretory pathway. These results are discussed in relation to the possible role of cyclophilins in protein folding and intracellular protein trafficking.

The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins.

Mutations in the Drosophila ninaA gene cause dramatic reductions in rhodopsin levels, leading to impaired visual function. The ninaA protein is a homolog of peptidyl-prolyl cis-trans isomerases. We find that ninaA is unique among this family of proteins in that it is an integral membrane protein, and it is expressed in a cell type-specific manner. We have used transgenic animals misexpressing different rhodopsins in the major class of photoreceptor cells to demonstrate that ninaA is required for normal function by two homologous rhodopsins, but not by a less conserved member of the Drosophila rhodopsin gene family. This demonstrates in vivo substrate specificity in a cyclophilin-like molecule. We also show that vertebrate retina contains a ninaA-related protein and that ninaA is a member of a gene family in Drosophila. These data offer insights into the in vivo role of this important family of proteins.

The ninaA gene required for visual transduction in Drosophila encodes a homologue of cyclosporin A-binding protein.

Mutations of the Drosophila melanogaster ninaA gene affect phototransduction: ninaA mutant flies have a 10-fold reduction in the levels of rhodopsin in the R1-R6 photoreceptor cells. The ninaA gene was isolated and found to encode a 237-amino-acid protein that has over 40% amino-acid sequence identity with the vertebrate cyclosporin A-binding protein, cyclophilin, a protein that seems to be involved in T-lymphocyte activation. The remarkable evolutionary conservation of cyclophilin in two phylogenetically distant organisms and its involvement in diverse transduction processes suggests that this protein plays an important role in cellular metabolism. Indeed, cyclophilin has recently been shown to be a prolyl cis-trans isomerase that catalyses, in vitro, rate-limiting steps in the folding of a number of proteins. Here, we present evidence for the involvement of cyclophilin-like molecules in a defined cellular process. The availability of mutations in a cyclophilin gene provides a new model system for the study of cyclophilin and cyclosporin action.

Cholera toxin and pertussis toxin substrates and endogenous ADP-ribosyltransferase activity in Drosophila melanogaster.

Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta. Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts. Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates. The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian Go and is influenced by guanyl nucleotide binding. The 39 kDa Go-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues. These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations.

Morphological and immunocytochemical observations on the visual callosal projections in the cat.

The connections between the left and right 17-18 border regions of the cat's visual cortex were labeled by axonal transport of peroxidase-conjugated wheat-germ agglutinin (WGA-HRP) and examined by light and electron microscopy. The cells of origin of the pathway were further characterized by transport of fluorescent microspheres ("beads") followed by in vitro injection of cells with Lucifer Yellow, and by beads transport followed by immunocytochemistry with antibodies to gamma-aminobutyric acid (GABA). The cells of origin of the callosal pathway were located in the lower part of layer 2/3, the upper part of layer 4, and layer 6. In layers 2/3 and 6, they were pyramidal cells; in layer 4 they were star pyramids or spiny stellate cells. None of them were spinefree or sparsely spinous cells, and none were GABA-positive. The axon terminals of the callosal pathway formed type 1 (asymmetric) synapses, and most of them contacted dendritic spines. Both the cells of origin and the terminals were arranged in patches. The findings suggest that the direct action of the callosal pathway is excitatory. The callosal system appears to represent only a subset of the cell types that have intrinsic horizontal projections within areas 17 or 18.